High glucose and homocysteine synergistically affect the metalloproteinases-tissue inhibitors of metalloproteinases pattern, but not TGFB expression, in human fibroblasts

Atherosclerosis is particularly aggressive in patients with diabetes. Hyperhomocysteinaemia causes oxidative stress and cytokine secretion: its atherogenic effect is mediated by an enhanced inflammatory response. Matrix metalloproteinases (MMPs) regulate extracellular matrix degradation and remodelling, and contribute to the vulnerability of the atherosclerotic lesion. Fibroblasts contribute to collagen biosynthesis and participate in plaque remodelling via expression and release of MMP2 and MMP9. To explore the role of hyperhomocysteinaemia in cellular pathways involved in plaque growth and stability in diabetic patients, we studied the effect of hyperhomocysteinaemia in human fibroblasts grown in the presence of normal or high glucose concentrations. In fibroblasts of five normal subjects, grown at 5.5 or 22 mmol/l glucose and treated with homocysteine, we determined: (1) MMP2, MMP9 and tissue inhibitor of metalloproteinases (TIMP)-1 (an MMP inhibitor) production by western blot analysis; (2) their activity by zymography; (3) TGFB1 expression by real-time PCR; and (4) TGFB, fibronectin and IL6 release by ELISA. Hyperhomocysteinaemia increased the production and enzymatic activity of MMP2 and MMP9, the effect being more pronounced in high glucose. Conversely, TIMP1 production was reduced by hyperhomocysteinaemia in both conditions, especially in high glucose. Hyperhomocysteinaemia also stimulated IL6 release, at least in part through nuclear factor-kappa B activation. TGFB1 expression was not affected by hyperhomocysteinaemia either in normal or in high glucose. Homocysteine upregulates the MMP-TIMP pathway and IL6 release, the effect being stronger in the presence of high glucose. These actions of homocysteine may contribute to the increased atherogenesis observed in diabetic patients with poor metabolic control.